The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energycoupling factor transporters for the new class of membrane transporters.Transport proteins residing in the cytoplasmic membrane allow the selective uptake and efflux of solutes and are essential for cellular growth and metabolism (20). Reflecting the importance of transporters, between 3% and 16% of the genes in prokaryote genomes are predicted to encode transporter proteins (26). These transporters form numerous families that are diverse in structure, energy-coupling mechanisms, and substrate specificities (25). As only a small fraction of predicted transporter proteins have known substrates, the functional prediction and annotation of the specificities of transporter proteins in the rapidly growing number of sequenced genomes represent a substantial challenge (25, 36). For example, the uptake of many cofactors and their precursors is essential for the growth of various pathogenic bacteria whose genomes are sequenced, but the transport proteins involved have not yet been identified. The use of computational comparative genomic techniques including gene colocalization, cooccurrence, and coregulation analyses combined with experimental assays is a powerful approach to identify novel transporters and to uncover their cellular role (for a recent review, see reference 11).The starting point for the present analysis was our recent discovery of multicomponent transport systems for the vitamin biotin (BioYNM) and the transition metals nickel (NikMNQO) and cobalt (CbiMNQO) (14,30). These transporters all have substrate-specific components (S components), which...
The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B 12 riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B 12 biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.The transition metals nickel and cobalt are essential cofactors for a number of prokaryotic enzymes involved in a variety of metabolic processes (36,41). Among the known nickeldependent enzymes are urease (8), [NiFe] hydrogenase, carbon monoxide dehydrogenase (Ni-CODH) (35), acetyl-coenzyme A decarbonylase/synthase (21), superoxide dismutase SodN (22), methyl-coenzyme M reductase (20), and glyoxylase I (50). In contrast to the diverse roles of nickel in microbial metabolism, cobalt is mainly found in the corrin ring of coenzyme B 12 , a cofactor involved in methyl group transfer and in rearrangement reactions (36). Since in natural environments, soluble Ni 2ϩ and Co 2ϩ are usually present only in trace amounts, the synthesis of the respective metalloenzymes requires high-affinity uptake of metal ions. Until recently, two major types of microbial high-affinity nickel and cobalt transporters were known: ATP-binding cassette (ABC) systems and secondary permeases of the NiCoT family (reviewed in reference 24).The NikABCDE system of Escherichia coli belongs to the nickel/peptide/opine ABC transporter family and is composed of the periplasmic binding protein NikA, two integral membrane components (NikB and -C), and two ATPases (NikD and -E) (43). The molecular basis of selective high-affinity binding of Ni 2ϩ remains elusive, although crystal structures of E. coli NikA have been determined by two approaches (10, 31).The two studies uncovered that N...
BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only onethird of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the ␣-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY- Biotin is synthesized by many bacteria, certain archaea, fungi, and plants (reviewed in ref. 2). Several metabolic routes seem to exist for the synthesis of the intermediate pimeloyl-CoA, which then is converted into biotin in a four-step path encoded by the universal genes bioF, bioA, bioD, and bioB (3, 4). In plants, the pathway is distributed between the cytosol and the mitochondria. At least the final step, catalyzed by biotin synthase, occurs in mitochondria. This enzyme, a member of the radical SAM enzyme family, inserts a sulfur atom into dethiobiotin in a complex reaction that is linked to mitochondrial iron/sulfur metabolism (2).Biotin uptake has been analyzed in eukaryotes. In mammalian cells, the vitamin is transported across the plasma membrane by a sodium-dependent multivitamin transporter and, at least in certain tissues, by monocarboxylate transporter 1 (1). In the naturally biotin-auxotrophic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, biotin uptake is mediated by unrelated proton symporters (5). Surprisingly little is known on the mechanisms behind biotin transport into prokaryotic cells, and multiple systems seem to exist. Active transport was observed for Escherichia coli K-12 Ͼ30 years ago (6). Despite extensive experimental work, knowledge of the complete genome sequence, and assignment of the biotin-transport locus to the 75-min genomic region, the gene(s) for the biotin transporter has not yet been identified. Recent studies by Walker and Altman (7) suggest that this system in E. coli and related Gram-negative bacteria not only transports the small vitamin, but in addition facilitates the uptake of biotinylated peptides with chain lengths up to 31 amino acid residues.In 2002, Entcheva et al. (8) reported that mutations in bioM and bioN lead to reduced biotin uptake in Sinorhizobium meliloti. Because the products of these two genes share distinct similarity with CbiO and CbiQ, which are components of prokary...
Nickel/cobalt permeases (NiCoTs, TC 2.A.52) are a rapidly growing family of structurally related membrane transporters whose members are found in Gram-negative and Gram-positive bacteria, in thermoacidophilic archaea, and in fungi. Previous studies have predicted two subclasses represented by HoxN of Ralstonia eutropha, a selective nickel transporter, and by NhlF of Rhodococcus rhodochrous, a nickel and cobalt transporter that displays a preference for the Co ion. In the present study, NiCoT genes of five Gram-negative bacteria and one Gram-positive bacterium were cloned and heterologously expressed in Escherichia coli. Based on substrate preference in metal-accumulation assays with the recombinant strains, two of the novel NiCoTs were assigned to the NhlF class. The remaining four NiCoTs belong to a yet unrecognized, third class. They transport both the nickel and the cobalt ion but have a significantly higher capacity for nickel. The observed substrate preferences correlate in many cases with the genomic localization of NiCoT genes adjacent to regions encoding nickel- or cobalt-dependent enzymes or enzymes involved in cobalamin biosynthesis. Alignment of 23 full-length NiCoT sequences and comparison with the available experimental data predict that substrate specificity of NiCoTs is an adaptation to specific transition metal requirements in various organisms from different taxa.
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